反义寡核苷酸抑制耐药基因逆转产ESBLs大肠埃希菌的多重耐药性

Reversal of multiple drug resistance in Escherichia coli by blocking CTX-M through antisense phosphothioate oligodeoxynucleotides

目的研究抑制产超广谱β-内酰胺酶（ESBLs）大肠埃希菌（Ecoli）耐药基因CTX-M表达的反义寡核苷酸（AS-ODNs）对其耐药性的影响。方法用脂质体包裹AS．ODNsW086后导人目的细菌B052，通过平板克隆形成实验计数菌落数（CFU）；微量法测定细菌生长曲线；通过RT．PCR法检测B052耐药基因CTX-M的表达变化，用液体稀释法检测W086对B052最小抑菌浓度（MIC）影响。结果AS—ODNsW086明显抑制B052的生长，W086各剂量组B052的CFU与空白对照组比较明显减少（P〈0．05），且具有浓度依赖性抑制效应，而单纯脂质体组、单纯W086组及对照链组与空白对照组无明显差异；W086能显著抑制B052CTX-M的表达。结论AS—ODNs能特异高效地与CTX—MmRNA结合并阻断其表达，有效逆转了B052的多重耐药性；AS-ODNs为解决细菌耐药问题提供了一个新的研究策略。

Objective To investigate the effects of antisense phosphothioate oligodeoxynucleotides （AS-ODNs） W086 on drug-resistant gene CTX-M expression in Escherichia coli producing extended-spectrum 13-lactamases （ESBLs）. Methods AS-ODNs liposome was introduced into the purpose bacteria [1052. The total colony forming unit（CFU） was counted. The bacteria growth curve was drawn by mieroplate reader. The inhibition effects of AS-ODNs on the expressions of drug-resistant gene CTX-M were observed by RT- PCR in B052. The minimal inhibitory concentration（ MIC）was determined by fluid dilution method. Results significant growth inhibition of cells treated with W086 was observed as compared with those in cells in con- trol treated bacteria. The number of B052 colonies significantly decreased in all W086 treated groups in a concentration dependent manner （ P 〈 0. 05 ）, while CFU of B052 was not influenced in simple liposome group, simple W086 group and controlled chain group. The expression of CTX-M was selectively inhibited. Conelnsion Efficiently and speeificly blocking expression of CTX-M mRNA, AS-ODNs reverses the multiple drug resistance of B052. It indicates that AS-ODNs provides a new viable strategy to reverse antibiotic resistance oroblem.